Rat GIP ELISA Assay

Additional Information

Assay Background

The incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagons-like peptide-1 (GLP-1), are a group of gastrointestinal hormones that cause an increase in the amount of insulin released from the beta cells of the islets of Langerhans after ingestion of food.
The intestinal peptide GIP was first isolated from porcine upper small intestine. The sequences of porcine, bovine and Rat GIP have been determined, each has 42 amino acids, and the sequences is highly conserved. The porcine and bovine peptides differ from the Rat at two and three site, respectively. Takeda et al. have isolated a Rat cDNA encoding the GIP precursor and confirming that GIP belongs to the vasoactive intestinal peptide (VIP)/Glucagon/secretin family. GIP is a gastrointestinal peptide hormone that is released from duodenal endocrine K cells after absorption of glucose or fat. GIP is a potent releaser of insulin in experimental animals and in man provided that the blood glucose is above basal level. Plasma level of GIP is elevated after an oral glucose load or a meal in normal man. This increase after a meal is below normal in newly diagnosed insulin–dependent diabetics. It is now being recognized that GIP receptor is also expressed in organs and cells such as duodenum, small intestine, pancreatic alpha-cell, adipocyte and osteoblast. These results demonstrate GIP may have a lot of physiological effect in addition to their glucoregulatory effects.
GIP is rapidly inactivated by the enzyme dipeptidyl peptidase- 4 (DPP- 4) to GIP (3-42) with a blood half-life of only several minutes. DPP- 4 inhibitor can prolong the half-life of GIP, that expecting treatment of incretin effect.
This ELISA kit has high specificity to rat GIP (1-42) active form and shows no cross reactivity to rat GIP (3-42) inactive form.

Assay Procedure

1. Before starting the assay, bring all the reagents and samples to room temperature (20 ~ 30ºC).
2. Fill 0.35 mL/well of washing solution into the wells and aspirate the washing solution in the wells. Repeat this washing procedure further twice (total 3 times). Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to ensure blotting free of most residual washing solution.
3. Add 50µL of buffer solution to the wells first, and then introduce 50µL of each of standard solutions (0, 3.9, 7.8, 15.6, 31.3, 62.5, 125 and 250 pg/mL) or samples to the wells.
4. Cover the plate with adhesive foil and incubate it at room temperature for 2 hours. During the incubation, the plate should be shaken with a plate shaker (approximately 100 rpm).
5. After incubation, take off the adhesive foil, aspirate and wash the wells 4 times with 0.35 mL/well of washing solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to ensure blotting free of most residual washing solution.
6. Add 100µL of HRP labeled antibody solution to each of the wells.
7. Cover the plate with adhesive foil and incubate it at room temperature for 1 hour. During the incubation, the plate should be shaken with a plate shaker (approximately 100 rpm).
8. Take off the adhesive foil, aspirate and wash the wells 4 times with 0.35 mL/well of washing solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to ensure blotting free of most residual washing solution.
9. Add 100µL of Enzyme substrate solution (TMB) to each of the wells, cover the plate with adhesive foil and keep it for 30 minutes at room temperature in a dark place for color reaction (keep still, plate shaker not need).
10. Add 100 µL of stopping solution to each of the wells to stop color reaction.
11. Read the optical absorbance of the solution in the wells at 450 nm. The dose-response curve of this assay fits best to a 5 (or 4)-parameter logistic equation. The results of unknown samples can be calculated with any computer program having a 5 (or 4)-parameter logistic function. Otherwise calculate mean absorbance values of wells containing standards and plot a standard curve on double logarithmic graph paper (abscissa: concentration of standard; ordinate: absorbance values). Use the average absorbance of each sample to determine the corresponding value by simple interpolation from this standard curve.

Typical Standard Curve